TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome.

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members, implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together, these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology.

A Gad2 specific Slc6a8 deletion recapitulates the contextual and cued freezing deficits seen in Slc6a8-/y mice.

The creatine (Cr)-phosphocreatine shuttle is essential for ATP homeostasis. In humans, the absence of brain Cr causes significant intellectual disability, epilepsy, and language delay. Mutations of the creatine transporter (SLC6A8) are the most common cause of Cr deficiency. In rodents, Slc6a8 deletion causes deficits in spatial learning, novel object recognition (NOR), as well as in contextual and cued freezing. The mechanisms that underlie these cognitive deficits are not known. Due to the heterogeneous nature of the brain, it is important to determine which systems are affected by a loss of Cr. In this study, we generated mice lacking Slc6a8 in GABAergic neurons by crossing Slc6a8FL mice with Gad2-Cre mice. These Gad2-specific Slc6a8 knockout (cKO) mice, along with the ubiquitous Slc6a8 KO (Slc6a8-/y), Gad2-Cre+, and wild-type (WT) mice were tested in the Morris water maze, NOR, conditioned freezing, and the radial water maze. Similar to the Slc6a8-/y mice, cKO mice had reduced contextual and cued freezing compared with WT mice. The cKO mice had a mild spatial learning deficit during the reversal phase of the MWM, however they were not as pronounced as in Slc6a8-/y mice. In NOR, the Gad2-Cre mice spent less time with the novel object, similar to the reduced novel time in the cKO mice. There were no changes in radial water maze performance. Slc6a8 deletion in GABAergic neurons is sufficient to recapitulate the conditioned freezing deficits seen in Slc6a8-/y mice.

H2A monoubiquitination: insights from human genetics and animal models.

Metazoan development arises from spatiotemporal control of gene expression, which depends on epigenetic regulators like the polycomb group proteins (PcG) that govern the chromatin landscape. PcG proteins facilitate the addition and removal of histone 2A monoubiquitination at lysine 119 (H2AK119ub1), which regulates gene expression, cell fate decisions, cell cycle progression, and DNA damage repair. Regulation of these processes by PcG proteins is necessary for proper development, as pathogenic variants in these genes are increasingly recognized to underly developmental disorders. Overlapping features of developmental syndromes associated with pathogenic variants in specific PcG genes suggest disruption of central developmental mechanisms; however, unique clinical features observed in each syndrome suggest additional non-redundant functions for each PcG gene. In this review, we describe the clinical manifestations of pathogenic PcG gene variants, review what is known about the molecular functions of these gene products during development, and interpret the clinical data to summarize the current evidence toward an understanding of the genetic and molecular mechanism.

Impulsive choice in two different rat models of ADHD-Spontaneously hypertensive and Lphn3 knockout rats.

Introduction: Impulsivity is a symptom of attention-deficit/hyperactivity disorder (ADHD) and variants in the Lphn3 (Adgrl3) gene (OMIM 616417) have been linked to ADHD. This project utilized a delay-discounting (DD) task to examine the impact of Lphn3 deletion in rats on impulsive choice. "Positive control" measures were also collected in spontaneously hypertensive rats (SHRs), another animal model of ADHD. Methods: For Experiment I, rats were given the option to press one lever for a delayed reward of 3 food pellets or the other lever for an immediate reward of 1 pellet. Impulsive choice was measured as the tendency to discount the larger, delayed reward. We hypothesized that impulsive choice would be greater in the SHR and Lphn3 knockout (KO) rats relative to their control strains - Wistar-Kyoto (WKY) and Lphn3 wildtype (WT) rats, respectively. Results: The results did not completely support the hypothesis, as only the SHRs (but not the Lphn3 KO rats) demonstrated a decrease in the percent choice for the larger reward. Because subsequent trials did not begin until the end of the delay period regardless of which lever was selected, rats were required to wait for the next trial to start even if they picked the immediate lever. Experiment II examined whether the rate of reinforcement influenced impulsive choice by using a DD task that incorporated a 1 s inter-trial interval (ITI) immediately after delivery of either the immediate (1 pellet) or delayed (3 pellet) reinforcer. The results of Experiment II found no difference in the percent choice for the larger reward between Lphn3 KO and WT rats, demonstrating reinforcement rate did not influence impulsive choice in Lphn3 KO rats. Discussion: Overall, there were impulsivity differences among the ADHD models, as SHRs exhibited deficits in impulsive choice, while the Lphn3 KO rats did not.

Developmental deltamethrin: Sex-specific hippocampal effects in Sprague Dawley rats.

Pyrethroid pesticides are widely used and can cause long-term effects after early exposure. Epidemiological and animal studies reveal associations between pyrethroid exposure and altered cognition following prenatal and/or neonatal exposure. However, little is known about the cellular effects of such exposure. Sprague Dawley rats were gavaged with 0 or 1.0 mg/kg deltamethrin (DLM), a Type II pyrethroid, in corn oil (dose volume 5 mL/kg) once per day from postnatal day (P) 3-20 and assessed shortly after dosing ended or as adults. No effects of DLM exposure were found on striatal dopaminergic markers, nor on AMPA receptor subunits or on NMDA-NR1. However, DLM increased NMDA-NR2A and decreased NMDA-NR2B levels in the hippocampus, in males but not females. Additionally, adult hippocampal CA1 long-term potentiation was increased in DLM-treated males but not females. Potassium stimulated extracellular glutamate release in the hippocampus was not affected using in vivo microdialysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed increased apoptotic cells in the dentate gyrus of male rats, in the absence of changes in cleaved caspase-3 at P21. Proinflammatory cytokines interferon gamma trended up in striatum, interleukin-1ß trended down in nucleus accumbens, IL-13 trended up in hippocampus, and keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO or CXCL1) was significantly increased in the hippocampus in male DLM-treated rats on P20. The data point to the developing hippocampus as a susceptible region to DLM-induced adverse effects.

Cognitive and behavioral effects of whole brain conventional or high dose rate (FLASH) proton irradiation in a neonatal Sprague Dawley rat model.

Recent studies suggest that ultra-high dose rates of proton radiation (>40 Gy/s; FLASH) confer less toxicity to exposed healthy tissue and reduce cognitive decline compared with conventional radiation dose rates (~1 Gy/s), but further preclinical data are required to demonstrate this sparing effect. In this study, postnatal day 11 (P11) rats were treated with whole brain irradiation with protons at a total dose of 0, 5, or 8 Gy, comparing a conventional dose rate of 1 Gy/s vs. a FLASH dose rate of 100 Gy/s. Beginning on P64, rats were tested for locomotor activity, acoustic and tactile startle responses (ASR, TSR) with or without prepulses, novel object recognition (NOR; 4-object version), striatal dependent egocentric learning ([configuration A] Cincinnati water maze (CWM-A)), prefrontal dependent working memory (radial water maze (RWM)), hippocampal dependent spatial learning (Morris water maze (MWM)), amygdala dependent conditioned freezing, and the mirror image CWM [configuration B (CWM-B)]. All groups had deficits in the CWM-A procedure. Weight reductions, decreased center ambulation in the open-field, increased latency on day-1 of RWM, and deficits in CWM-B were observed in all irradiated groups, except the 5 Gy FLASH group. ASR and TSR were reduced in the 8 Gy FLASH group and day-2 latencies in the RWM were increased in the FLASH groups compared with controls. There were no effects on prepulse trials of ASR or TSR, NOR, MWM, or conditioned freezing. The results suggest striatal and prefrontal cortex are sensitive regions at P11 to proton irradiation, with reduced toxicity from FLASH at 5 Gy.

Latrophilin-3 heterozygous versus homozygous mutations in Sprague Dawley rats: Effects on egocentric and allocentric memory and locomotor activity.

Latrophilin-3 (LPHN3) is a brain specific G-protein coupled receptor associated with increased risk of attention deficit hyperactivity disorder (ADHD) and cognitive deficits. CRISPR/Cas9 was used to generate a constitutive knockout (KO) rat of Lphn3 by deleting exon 3, based on human data that LPHN3 variants are associated with some cases of ADHD. Lphn3 KO rats are hyperactive with an attenuated response to ADHD medication and have cognitive deficits. Here, we tested KO, heterozygous (HET), and wildtype (WT) rats to determine if there was a gene-dosage effect. We tested the rats in home-cage activity starting at postnatal day (P)35 and P50, followed by tests of egocentric learning (Cincinnati water maze [CWM]), spatial learning (Morris water maze [MWM]), working memory (radial water maze [RWM]), incidental learning (novel object recognition [NOR]), acoustic startle response (ASR) habituation, tactile startle response (TSR) habituation, prepulse modification of acoustic startle, shuttle-box passive avoidance, conditioned freezing, and a mirror image version of the CWM. KO and HET rats were hyperactive. KO and HET rats had egocentric (CWM) and spatial deficits (MWM), increased startle response, and KO rats showed less conditioned freezing on contextual and cued memory; there were no effects on working memory (RWM) or passive avoidance. The selective gene-dosage effect in Lphn3 HET rats indicates that Lphn3 exhibits dominate expression on functions where it is most abundantly expressed (striatum, hippocampus) but not on behaviors mediated by regions of low expression. The data add further evidence to the impact of this synaptic protein on brain function and behavior.

Review of rodent models of attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is a polygenic neurodevelopmental disorder that affects 8-12 % of children and >4 % of adults. Environmental factors are believed to interact with genetic predispositions to increase susceptibility to ADHD. No existing rodent model captures all aspects of ADHD, but several show promise. The main genetic models are the spontaneous hypertensive rat, dopamine transporter knock-out (KO) mice, dopamine receptor subtype KO mice, Snap-25 KO mice, guanylyl cyclase-c KO mice, and latrophilin-3 KO mice and rats. Environmental factors thought to contribute to ADHD include ethanol, nicotine, PCBs, lead (Pb), ionizing irradiation, 6-hydroxydopamine, neonatal hypoxia, some pesticides, and organic pollutants. Model validation criteria are outlined, and current genetic models evaluated against these criteria. Future research should explore induced multiple gene KOs given that ADHD is polygenic and epigenetic contributions. Furthermore, genetic models should be combined with environmental agents to test for interactions.

A novel role for the ADHD risk gene latrophilin-3 in learning and memory in Lphn3 knockout rats.

Latrophilins (LPHNs) are adhesion G protein-coupled receptors with three isoforms but only LPHN3 is brain specific (caudate, prefrontal cortex, dentate, amygdala, and cerebellum). Variants of LPHN3 are associated with ADHD. Null mutations of Lphn3 in rat, mouse, zebrafish, and Drosophila result in hyperactivity, but its role in learning and memory (L&M) is largely unknown. Using our Lphn3 knockout (KO) rats we examined the cognitive abilities, long-term potentiation (LTP) in CA1, NMDA receptor expression, and neurohistology from heterozygous breeding pairs. KO rats were impaired in egocentric L&M in the Cincinnati water maze, spatial L&M and cognitive flexibility in the Morris water maze (MWM), with no effects on conditioned freezing, novel object recognition, or temporal order recognition. KO-associated locomotor hyperactivity had no effect on swim speed. KO rats had reduced early-LTP but not late-LTP and had reduced hippocampal NMDA-NR1 expression. In a second experiment, KO rats responded to a light prepulse prior to an acoustic startle pulse, reflecting visual signal detection. In a third experiment, KO rats given extra MWM pretraining and hidden platform overtraining showed no evidence of reaching WT rats' levels of learning. Nissl histology revealed no structural abnormalities in KO rats. LPHN3 has a selective effect on egocentric and allocentric L&M without effects on conditioned freezing or recognition memory.

An assessment of executive function in two different rat models of attention-deficit hyperactivity disorder: Spontaneously hypertensive versus Lphn3 knockout rats.

Attention-deficit/hyperactivity disorder (ADHD) a common neurodevelopmental disorder of childhood and often comorbid with other externalizing disorders (EDs). There is evidence that externalizing behaviors share a common genetic etiology. Recently, a genome-wide, multigenerational sample linked variants in the Lphn3 gene to ADHD and other externalizing behaviors. Likewise, limited research in animal models has provided converging evidence that Lphn3 plays a role in EDs. This study examined the impact of Lphn3 deletion (i.e., Lphn3-/- ) in rats on measures of behavioral control associated with externalizing behavior. Impulsivity was assessed for 30 days via a differential reinforcement of low rates (DRL) task and working memory evaluated for 25 days using a delayed spatial alternation (DSA) task. Data from both tasks were averaged into 5-day testing blocks. We analyzed overall performance, as well as response patterns in just the first and last blocks to assess acquisition and steady-state performance, respectively. "Positive control" measures on the same tasks were measured in an accepted animal model of ADHD-the spontaneously hypertensive rat (SHR). Compared with wildtype controls, Lphn3-/- rats exhibited deficits on both the DRL and DSA tasks, indicative of deficits in impulsive action and working memory, respectively. These deficits were less severe than those in the SHRs, who were profoundly impaired on both tasks compared with their control strain, Wistar-Kyoto rats. The results provide evidence supporting a role for Lphn3 in modulating inhibitory control and working memory, and suggest additional research evaluating the role of Lphn3 in the manifestation of EDs more broadly is warranted.

Latrophilin-3 disruption: Effects on brain and behavior.

Latrophilin-3 (LPHN3), a G-protein-coupled receptor belonging to the adhesion subfamily, is a regulator of synaptic function and maintenance in brain regions that mediate locomotor activity, attention, and memory for location and path. Variants of LPHN3 are associated with increased risk for attention deficit hyperactivity disorder (ADHD) in some patients. Here we review the role of LPHN3 in the central nervous system (CNS). We describe synaptic localization of LPHN3, its trans-synaptic binding partners, links to neurodevelopmental disorders, animal models of Lphn3 disruption in different species, and evidence that LPHN3 is involved in cognition as well as activity and attention. The evidence shows that LPHN3 plays a more significant role in neuroplasticity than previously appreciated.

Effects of Permethrin or Deltamethrin Exposure in Adult Sprague Dawley Rats on Acoustic and Light Prepulse Inhibition of Acoustic or Tactile Startle.

The effects of permethrin (PRM) and deltamethrin (DLM) on acoustic or light prepulse inhibition of the acoustic startle response (ASR) and tactile startle response (TSR) were studied in adult male Sprague Dawley rats. Preliminary studies were conducted to optimize the parameters of light and acoustic prepulse inhibition of ASR and TSR. Once these parameters were set, a new group of rats was administered PRM (0 or 90 mg/kg) or DLM (0 or 25 mg/kg) by gavage in 5 mL/kg corn oil. ASR and TSR were assessed using acoustic or light prepulses 6, 8, and 12 h after PRM and 2, 4, and 6 h after DLM exposure. PRM increased ASR 6 h post-treatment with no interaction with acoustic prepulse levels and with no effect on TSR. When light was used as the prepulse, PRM increased ASR and TSR at 6 h with no interaction with prepulse levels. DLM decreased ASR and TSR on trials without prepulses but not on trials with acoustic prepulses. DLM also decreased ASR when light prepulses were present 4 h post-treatment. A final experiment assessed whether the house light in the test cabinet affected ASR and TSR after PRM or DLM exposure. Rats had increased ASR and TSR when house lights were on compared with when they were off, but lighting did not differentially interact with PRM or DLM. Light and acoustic prepulses of ASR and TSR have different effects depending on the test agent and the test parameters.

Whole brain proton irradiation in adult Sprague Dawley rats produces dose dependent and non-dependent cognitive, behavioral, and dopaminergic effects.

Proton radiotherapy causes less off-target effects than X-rays but is not without effect. To reduce adverse effects of proton radiotherapy, a model of cognitive deficits from conventional proton exposure is needed. We developed a model emphasizing multiple cognitive outcomes. Adult male rats (10/group) received a single dose of 0, 11, 14, 17, or 20 Gy irradiation (the 20 Gy group was not used because 50% died). Rats were tested once/week for 5 weeks post-irradiation for activity, coordination, and startle. Cognitive assessment began 6-weeks post-irradiation with novel object recognition (NOR), egocentric learning, allocentric learning, reference memory, and proximal cue learning. Proton exposure had the largest effect on activity and prepulse inhibition of startle 1-week post-irradiation that dissipated each week. 6-weeks post-irradiation, there were no effects on NOR, however proton exposure impaired egocentric (Cincinnati water maze) and allocentric learning and caused reference memory deficits (Morris water maze), but did not affect proximal cue learning or swimming performance. Proton groups also had reduced striatal levels of the dopamine transporter, tyrosine hydroxylase, and the dopamine receptor D1, effects consistent with egocentric learning deficits. This new model will facilitate investigations of different proton dose rates and drugs to ameliorate the cognitive sequelae of proton radiotherapy.

Subsecond spontaneous catecholamine release in mesenteric lymph node ex vivo.

Measuring the dynamics of neurochemical-regulated immunity, particularly in the gut, has been a growing interest over the last several years because of its important implications in gastrointestinal inflammation, neurodegeneration, and even depression. Sympathetic noradrenergic nerves innervate the gastrointestinal tract and resident immune organs, including the mesenteric lymph nodes (MLN) and Peyer's patches. Previous research has suggested that neuronal inputs in the MLN release norepinephrine (NE) at neural-immune synapses to regulate immune function. The current immunological techniques do not have the appropriate temporal or spatial resolution to monitor this dynamic process in real-time, within specific regions of intact lymphoid organs. Monitoring dynamic neural signaling within intact immune organs, in real-time, would facilitate a deeper understanding of neuroimmune communication and would allow the mechanism of rapid immunomodulation to be elucidated. Here, we overcome this technological barrier by coupling real-time neurochemical detection using fast-scan cyclic voltammetry (FSCV) in live MLN slices from C57BL/6 mice. We have discovered rapid, spontaneous catecholamine transients in the T-cell zone of the MLN which are on the order of a few hundred nanomolar, rapid (a few seconds), and frequent (every 20-s). We demonstrate that the ß2 -adrenergic receptor and the classic catecholamine transporters (DAT and NET) play a minor role in transient regulation in the MLN suggesting that regulation at the neural-immune synapse is quite complicated and further mechanistic studies are needed. Overall, these findings provide direct evidence for rapid neurochemical events in the MLN which could have a major impact on our understanding of neurochemical-regulated immunomodulation in the gut.

Enhanced Transient Striatal Dopamine Release and Reuptake in Lphn3 Knockout Rats.

Latrophilin-3 (LPHN3) is an adhesion G protein coupled receptor involved in regulating neuroplasticity. Variants of LPHN3 are associated with increased risk of attention-deficit hyperactivity disorder. Data from mouse, zebrafish, Drosophila, and rat show that disruption of LPHN3 results in hyperactivity, and in the Sprague-Dawley Lphn3 knockout rat, exhibit deficits in learning and memory and changes in dopamine (DA) markers in the neostriatum. To determine the effects of Lphn3 deletion on DA neurotransmission, we compared the concentration, duration, and frequency of DA transients in KO and wild-type rats using fast-scan cyclic voltammetry in brain slices. Lphn3 KO rats showed higher release of DA, and the duration and interevent time were markedly decreased compared with wild-type rats. The data demonstrate that LPHN3 plays a heretofore unrecognized role in DA signaling and may represent a new target for small molecule regulation of DA neurotransmission with translational implications.

Knockout of latrophilin-3 in Sprague-Dawley rats causes hyperactivity, hyper-reactivity, under-response to amphetamine, and disrupted dopamine markers.

Attention deficit hyperactivity disorder is a pervasive developmental disorder characterized by inattention, impulsivity, and hyperactivity and is 75-90% heritable. Latrophilin-3 (LPHN3; or ADGRL(3)) is associated with a subtype of ADHD, but how it translates to symptoms is unknown. LPHN3 is a synaptic adhesion G protein coupled receptor that binds to fibronectin leucine rich transmembrane protein 3 and teneurin-3 (FLRT3 and TEN-3). We created a null mutation of Lphn3 (KO) in Sprague-Dawley rats using CRISPR/Cas9 to delete exon-3. The KO rats had no effects on reproduction or survival but reduced growth. KO females showed catch-up weight gain whereas KO males did not. We tested WT and KO littermates for home-cage activity, anxiety-like behavior, acoustic startle response, and activity after amphetamine challenge. Expression of Lphn3-related genes, monoamines, and receptors were determined. Lphn3 KO rats showed persistent hyperactivity, increased acoustic startle, reduced activity in response to amphetamine relative to baseline, and female-specific reduced anxiety-like behavior. Expression of Lphn1, Lphn2, and Flrt3 by qPCR and their protein products by western-blot analysis showed no compensatory upregulation. Striatal tyrosine hydroxylase, aromatic L-amino acid decarboxylase (AADC), and the dopamine transporter were increased and dopamine D1 receptor (DRD1) and dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) decreased with no changes in DRD2, DRD4, vesicular monoamine transporter-2, N-methyl-d-aspartate (NMDA)-NR1, -NR2A, or -NR2B. LPHN3 is expressed in many brain regions but its function is largely unknown. Data from human, mouse, zebrafish, Drosophila and our new Lphn3 KO rat data collectively show that its disruption is significantly correlated with hyperactivity and associated striatal changes in dopamine markers.

Effects of intrastriatal dopamine D1 or D2 antagonists on methamphetamine-induced egocentric and allocentric learning and memory deficits in Sprague-Dawley rats.

RATIONALE: Methamphetamine (MA) is an abused psychostimulant that causes cognitive deficits after chronic use. Neostriatal dopamine receptors play a role in MA monoamine neurotoxicity. Blocking dopamine receptors prior to MA exposure in adult rats attenuates monoamine reductions and reactive gliosis. OBJECTIVES: We tested whether blocking dopamine receptors protects against cognitive deficits. METHODS: First, we determined the effects of MA alone versus MA in combination with the dopamine receptor D1 antagonist SCH-23390 or the dopamine receptor D2 antagonist sulpiride on cFos expression and monoamines at the age when rats in the cognitive experiment were to begin testing and monoamines in rats after cognitive testing. RESULTS: SCH-23390 infused into the neostriatum prior to systemic administration of MA attenuated MA-induced cFos activation while sulpiride induced cFos activation. Two weeks after MA, rats had dopamine and serotonin reductions that were attenuated by each antagonist. Other rats treated the same way, were tested for egocentric learning and memory in the Cincinnati water maze, for navigational strategy in a star water maze, and spatial learning and memory in a Morris water maze. Pre-treatment with SCH-23390 or sulpiride attenuated the effects of MA on egocentric and spatial learning and memory. MA-treated rats showed a shift from an egocentric to a disorganized strategy in the star maze that was less disorganized in groups receiving MA and an antagonist. Post-behavior monoamine reductions remained but were attenuated by the antagonists but not identically to what was seen in rats not behaviorally tested. CONCLUSIONS: The results show for the first time that dopamine receptors are mediators of MA-induced cognitive deficits.

A better approach to in vivo developmental neurotoxicity assessment: Alignment of rodent testing with effects seen in children after neurotoxic exposures.

High throughput screens for developmental neurotoxicity (DN) will facilitate evaluation of chemicals and can be used to prioritize those designated for follow-up. DN is evaluated under different guidelines. Those for drugs generally include peri- and postnatal studies and juvenile toxicity studies. For pesticides and commercial chemicals, when triggered, include developmental neurotoxicity studies (DNT) and extended one-generation reproductive toxicity studies. Raffaele et al. (2010) reviewed 69 pesticide DNT studies and found two of the four behavioral tests underperformed. There are now many epidemiological studies on children showing adverse neurocognitive effects, yet guideline DN studies fail to assess most of the functions affected in children; nor do DN guidelines reflect the advances in brain structure-function relationships from neuroscience. By reducing the number of test ages, removing underperforming tests and replacing them with tests that assess cognitive abilities relevant to children, the value of DN protocols can be improved. Testing for the brain networks that mediate higher cognitive functions need to include assessments of working memory, attention, long-term memory (explicit, implicit, and emotional), and executive functions such as cognitive flexibility. The current DNT focus on what can be measured should be replaced with what should be measured. With the wealth of data available from human studies and neuroscience, the recommendation is made for changes to make DN studies better focused on human-relevant functions using tests of proven validity that assess comparable functions to tests used in children. Such changes will provide regulatory authorities with more relevant data.